1: Leukemia. 2005 Nov;19(11):1958-68. Effects of overexpression of HBP1 upon growth and differentiation of leukemic myeloid cells. Yao CJ, Works K, Romagnoli PA, Austin GE. Department of Pathology and Laboratory Medicine, Veterans Affairs Medical Center, Decatur, GA 30033, USA. HMG-box containing protein 1 (HBP1) is a member of the high mobility group (HMG) of chromosomal proteins. Since HBP1 exhibits tumor-suppressor activity in nonmyeloid tissues, we examined the effects of ectopic overexpression of HBP1 upon the growth and differentiation of myeloid cells. We prepared transient and stable transfectants of the myeloblast cell line K562, which overexpress HBP1 mRNA and protein. HBP1 transfectants displayed slower growth in cell culture and reduced colony formation in soft agar, retardation of S-phase progression, reduced expression of cyclin D1 and D3 mRNAs and increased expression of p21 mRNA. HBP1 transfectants also underwent increased apoptosis, as demonstrated by morphology and binding of Annexin V. Fas ligand mRNA levels were increased in HBP1 transfectants, suggesting involvement of the Fas/Fas ligand pathway. HBP1 overexpression enhanced differentiation of K562 cells towards erythroid and megakaryocyte lineages, as evidenced by increased hemoglobin and CD41a expression. Overexpression of HBP1 modulated mRNA levels for myeloid-specific transcription factors C/EBPalpha, c-Myb, c-Myc, and JunB, as well as lineage-specific transcription factors PU.1, GATA-1, and RUNX1. These findings suggest that in myeloid cells HBP1 may serve as a tumor suppressor and a general differentiation inducer and may synergize with chemical differentiating agents to enhance lineage-specific differentiation. PMID: 16179914 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Gene Expr Patterns. 2005 Jul 18; [Epub ahead of print] Core binding factor in the early avian embryo: cloning of Cbfbeta and combinatorial expression patterns with Runx1. Bollerot K, Romero S, Dunon D, Jaffredo T. UMR7622, UMPC Paris VI, 9, Quai St Bernard, 75005 Paris, France. We have isolated the avian ortholog for CBFbeta, the common non-DNA binding subunit of the core binding factor (CBF) that has important regulatory roles in major developmental pathways. CBFbeta forms heterodimers with the DNA-binding Runx proteins and increases their affinity for DNA and their protein stability. Here, we describe the Cbfbeta expression pattern during the first 4 days of chick embryo development, with a special interest in the developing hematopoietic system. We have compared its expression pattern to that of Runx1, which is crucial for the generation of definitive hematopoietic cells, and to other hematopoietic- or endothelial-specific markers (c-Myb, Pu.1, CD45, c-Ets-1 and VE-Cadherin). Initially, Cbfbeta is widely expressed in the early mesoderm in both the yolk sac and the embryo proper, but later its expression becomes restricted to specific organs or cell types. We have found that Cbfbeta expression overlaps with Runx1 in the hematopoietic system and neural tube. The somitic and mesonephric structures, however, express Cbfbeta in the absence of detectable Runx1. Finally, Cbfbeta and Runx1 display multiple combinatorial patterns in the endoderm and in specific nerves or ganglia. Taken together, we show that Cbfbeta exhibits a dynamic expression pattern that varies according to the organ, cell type or developmental stage. By revealing multiple combinatorial patterns between Cbfbeta and Runx1, these data provide new insights into the role of CBF during early development. PMID: 16033710 [PubMed - as supplied by publisher] --------------------------------------------------------------- 3: J Immunol. 2005 Apr 1;174(7):4144-52. Regulation of the murine Ddelta2 promoter by upstream stimulatory factor 1, Runx1, and c-Myb. Carabana J, Ortigoza E, Krangel MS. Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA. Accessibility control of V(D)J recombination at Ag receptor loci depends on the coordinate activities of transcriptional enhancers and germline promoters. Recombination of murine Tcrd gene segments is known to be regulated, at least in part, by the Tcrd enhancer (Edelta) situated in the Jdelta2-Cdelta intron. However, there has been little characterization of promoters and other cis-acting elements that are activated by or collaborate with Edelta and that might function to regulate Tcrd gene recombination events. We now describe a strong promoter that is tightly associated with the murine Ddelta2 gene segment. EMSAs reveal that upstream stimulatory factor 1, Runx1, c-Myb, lymphoid enhancer binding factor 1, NF1, and E47 all interact with this promoter in vitro. Of these, upstream stimulatory factor 1, Runx1, and c-Myb appear necessary for full promoter activity in transiently transfected cells. Moreover, the same three factors were found to interact with the promoter in vivo by chromatin immunoprecipitation. We suggest that these factors play important roles as Edelta-dependent regulators of Ddelta2 accessibility in vivo. Consistent with the established roles of c-Myb and Runx factors in Edelta function, we detected low level, enhancer-independent activity of the Ddelta2 promoter in transient transfection experiments. We speculate that the Ddelta2 promoter may play a role as a weak, enhancer-independent regulator in vivo, and might contribute to residual Tcrd rearrangement in Edelta(-/-) mice. PMID: 15778374 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Seikagaku. 2004 Oct;76(10):1305-19. [Biochemistry and structural biology in the post-genome era: search for the underlying mechanisms of biological molecules] [Article in Japanese] Ogata K. Department of Biochemistry/Gene Regulation, Yokohama City University Graduate School of Medicine, 3 9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Publication Types: Review Review, Tutorial PMID: 15580862 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Curr Opin Struct Biol. 2003 Feb;13(1):40-8. Erratum in: Curr Opin Struct Biol. 2003 Apr;13(2):262. Tahirov Tahir [corrected to Tahirov Tahir H]. Eukaryotic transcriptional regulatory complexes: cooperativity from near and afar. Ogata K, Sato K, Tahirov TH. Department of Biochemistry, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. ogata@med.yokohama-cu.ac.jp It is characteristic of eukaryotic transcription that a unique combination of multiple transcriptional regulatory proteins bound to promoter DNA specifically activate or repress downstream target genes; this is referred to as combinatorial gene regulation. Recently determined structures have revealed different modes of protein-protein interaction on the promoter DNA from near (e.g. the Runx1-CBFbeta-DNA, NFAT-Fos-Jun-DNA, GABPalpha-GABPbeta-DNA, Ets-1-Pax-5-DNA and PU.1-IRF-4-DNA complexes) and afar with DNA looping (e.g. the c-Myb-C/EBPbeta-DNA complex), and their regulatory mechanisms. Publication Types: Review Review, Tutorial PMID: 12581658 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Blood. 2003 Feb 1;101(3):886-93. Epub 2002 Sep 19. Hemogenic and nonhemogenic endothelium can be distinguished by the activity of fetal liver kinase (Flk)-1 promoter/enhancer during mouse embryogenesis. Hirai H, Ogawa M, Suzuki N, Yamamoto M, Breier G, Mazda O, Imanishi J, Nishikawa S. Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, Japan. hhirai@basic.kpu-m.ac.jp Accumulating evidence in various species has suggested that the origin of definitive hematopoiesis is associated with a special subset of endothelial cells (ECs) that maintain the potential to give rise to hematopoietic cells (HPCs). In this study, we demonstrated that a combination of 5'-flanking region and 3' portion of the first intron of the Flk-1 gene (Flk-1 p/e) that has been implicated in endothelium-specific gene expression distinguishes prospectively the EC that has lost hemogenic activity. We assessed the activity of this Flk-1 p/e by embryonic stem (ES) cell differentiation culture and transgenic mice by using the GFP gene conjugated to this unit. The expression of GFP differed from that of the endogenous Flk-1 gene in that it is active in undifferentiated ES cells and inactive in Flk-1(+) lateral mesoderm. Flk-1 p/e becomes active after generation of vascular endothelial (VE)-cadherin(+) ECs. Emergence of GFP(-) ECs preceded that of GFP(+) ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell sorting experiments demonstrated that only GFP(-) ECs could give rise to HPCs and preferentially expressed Runx1 and c-Myb genes that are required for the definitive hematopoiesis. Integration of both GFP(+) and GFP(-) ECs was observed in the dorsal aorta, but cell clusters appeared associated only to GFP(-) ECs. These results indicate that activation of Flk-1 p/e is associated with a process that excludes HPC potential from the EC differentiation pathway and will be useful for investigating molecular mechanisms underlying the divergence of endothelial and hematopoietic lineages. PMID: 12393724 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Cell Biochem. 2002;86(4):624-9. Runx1, c-Myb, and C/EBPalpha couple differentiation to proliferation or growth arrest during hematopoiesis. Friedman AD. Department of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland 21231, USA. afriedm2@jhem.jhmi.edu Immature hematopoietic precursors proliferate as they differentiate, whereas terminal differentiation is associated with cell cycle arrest. Stem cell lineage commitment and subseqent maturation is regulated predominantly by transcription factors. Runx1 and c-Myb act in early stage hematopoietic cells to both stimulate proliferation and differentiation, whereas C/EBPalpha, and perhaps other C/EBP family members, block progression from G1 to S and induce terminal maturation. Coupling of differentiation to either proliferation or growth arrest by transcription factors is likely an important regulatory mechanism in multiple developmental systems. Copyright 2002 Wiley-Liss, Inc. Publication Types: Review Review, Tutorial PMID: 12210729 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Int Rev Immunol. 2001 Feb;20(1):83-105. Transcription factors that regulate growth and differentiation of myeloid cells. Nagamura-Inoue T, Tamura T, Ozato K. Laboratory of Molecular Growth Regulation National Institute of Child Health and Human Development National Institutes of Health, Bethesda, MD 20892-2753, USA. Recently much progress has been made in our understanding of how myeloid progenitor cells undergo commitment and become mature granulocytes or monocytes/macrophages. Studies of normal and leukemic myeloid cells as well as those of cells derived from mice with targeted disruption showed that a series of transcription factors play a major role in both commitment and maturation of myeloid cells. This is primarily because these transcription factors direct an ordered pattern of gene expression according to a well-defined developmental program. PU.1, an Ets family member, is one of the master transcription factors identified to regulate development of both granulocytes and monocytes/macrophages. Further, C/EBPalpha and C/EBPvarepsilon of the bZip family have important roles in directing granulocytic maturation. A number of additional transcription factors such as AML1, RARalpha, MZF-1, Hox and STAT families of transcription factors, Egr-1 and c-myb etc are shown to play roles in myeloid cell differentiation. Our laboratory has recently obtained evidence that ICSBP, a member of the IRF family, is involved in lineage commitment during myeloid cell differentiation and stimulates maturation of functional macrophages. Future elucidation of pathways and networks through which these transcription factors act in various stages of development would provide a more definitive picture of myeloid cell commitment and maturation. Publication Types: Review PMID: 11342299 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: J Virol. 2001 Mar;75(5):2174-84. Type B leukemogenic virus has a T-cell-specific enhancer that binds AML-1. Mertz JA, Mustafa F, Meyers S, Dudley JP. Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA. Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell tumors in mice. TBLV is highly related to mouse mammary tumor virus (MMTV) except that TBLV long terminal repeats (LTRs) have a deletion of negative regulatory elements and a triplication of sequences flanking the deletion. To determine if the LTR triplication represents a viral enhancer element, we inserted the triplication upstream and downstream in either orientation relative to the thymidine kinase promoter linked to the luciferase gene. These experiments showed that upregulation of reporter gene activity by the TBLV triplication was relatively orientation independent, consistent with the activity of eukaryotic enhancer elements. TBLV enhancer activity was observed in T-cell lines but not in fibroblasts, B cells, or mammary cells, suggesting that enhancer function is cell type dependent. To analyze the transcription factor binding sites that are important for TBLV enhancer function, we prepared substitution mutations in a reconstituted C3H MMTV LTR that recapitulates the deletion observed in the TBLV LTR. Transient transfections showed that a single mutation (556M) decreased TBLV enhancer activity at least 20-fold in two different T-cell lines. This mutation greatly diminished AML-1 (recently renamed RUNX1) binding in gel shift assays with a mutant oligonucleotide, whereas AML-1 binding to a wild-type TBLV oligomer was specific, as judged by competition and supershift experiments. The 556 mutation also reduced TBLV enhancer binding of two other protein complexes, called NF-A and NF-B, that did not appear to be related to c-Myb or Ets. AML-1 overexpression in a mammary cell line enhanced expression from the TBLV LTR approximately 30-fold. These data suggest that binding of AML-1 to the TBLV enhancer, likely in combination with other factors, is necessary for optimal enhancer function. PMID: 11160721 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Dev Biol. 2000 Apr 1;220(1):27-36. The AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic aorta-gonad-mesonephros region. Mukouyama Y, Chiba N, Hara T, Okada H, Ito Y, Kanamaru R, Miyajima A, Satake M, Watanabe T. Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113, Japan. We examined the role of the AML1 transcription factor in the development of hematopoiesis in the paraaortic splanchnopleural (P-Sp) and the aorta-gonad-mesonephros (AGM) regions of mouse embryos. The activity of colony-forming units of colonies from the P-Sp/AGM region was reduced severalfold by heterozygous disruption of the AML1 gene, indicating that AML1 functioned in a dosage-dependent manner to generate hematopoietic progenitors. In addition, no hematopoietic progenitor activity was detected in the P-Sp/AGM region of embryos with an AML1 null mutation. Similar results were obtained when a dispersed culture was first prepared from the P-Sp/AGM region before assay of the activity of the colony-forming units. In a culture of cells with the AML1(+/+) genotype, both hematopoietic and endothelial-like cell types emerged, but in a culture of cells with the AML1(-/-) genotype, only endothelial-like cells emerged. Interestingly, introduction of AML1 cDNA into the P-Sp/AGM culture with the AML1(-/-) genotype partially restored the production of hematopoietic cells. This restoration was observed for cultures prepared from 9.5-day postcoitum (dpc) embryos but not for cultures prepared from 11.5-dpc embryos. Therefore, the population of endothelial-like cells capable of growing in the AML1(-/-) culture would appear to contain inert but nonetheless competent hematogenic precursor cells up until at least the 9.5-dpc period. All these results support the notion that the AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic P-Sp/AGM region. Copyright 2000 Academic Press. PMID: 10720428 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Blood. 2000 Feb 1;95(3):745-55. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blobel GA. Division of Hematology, Children's Hospital of Philadelphia, and the University of Pennsylvania School of Medicine, Philadelphia, PA, USA. Publication Types: Review PMID: 10648382 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Oncogene. 1998 Nov 5;17(18):2287-93. AML1(-/-) embryos do not express certain hematopoiesis-related gene transcripts including those of the PU.1 gene. Okada H, Watanabe T, Niki M, Takano H, Chiba N, Yanai N, Tani K, Hibino H, Asano S, Mucenski ML, Ito Y, Noda T, Satake M. Department of Cell Biology, Cancer Institute, Tokyo, Japan. The AML1 and PEBP2beta/CBFbeta genes encode the DNA-binding and non-binding subunits, respectively, of the heterodimeric transcription factor, PEBP2/CBF. Targeting each gene results in an almost identical phenotype, namely the complete lack of definitive hematopoiesis in the fetal liver on embryonic day 11.5 (E11.5). We examined and compared the expression levels of various hematopoiesis-related genes in wild type embryos and in embryos mutated for AML1 or PEBP2beta/CBFbeta. The RNAs were prepared from the yolk sacs of E9.5 embryos, from the aorta-gonad- mesonephros regions of E11.5 embryos and from the livers of E11.5 embryos and RT-PCR was performed to detect various gene transcripts. Transcripts were detected for most of the hematopoiesis-related genes that encode transcription factors, cytokines and cytokine receptors, even in tissues from homozygously targeted embryos. On the other hand, PU.1 transcripts were never detected in any tissue of AML1(-/-) or PEBP2beta/CBFbeta(-/-) embryos. In addition, transcripts for the Vav, flk-2/flt-3, M-CSF receptor, G-CSF receptor and c-Myb genes were not detected in certain tissues of the (-/-) embryos. The results suggest that the expression of a particular set of hematopoiesis-related genes is closely correlated with the PEBP2/CBF function. PMID: 9811459 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1935-40. Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions. Ghozi MC, Bernstein Y, Negreanu V, Levanon D, Groner Y. Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel. The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1. PMID: 8700862 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------